A practical application of single human rotavirus gene substitution reassortants is found in the study of genetic relatedness of the genes coding for VP7 of human rotaviruses belonging to serotypes 1 through 4. Double-stranded (ds) genomic RNAs of human rotaviruses belonging to serotypes 1-4 were hybridized to single stranded (ss) mRNA probes derived from human-bovine rotavirus reassortants containing only the VP7 gene of their human rotavirus parent. Bovine rotavirus genes do not hybridize to the corresponding genes of human rotaviruses and thus hybridization can only occur between the human VP7 gene in the labeled probe and the VP7 gene present in the human rotavirus under study. A high degree of homology was demonstrated between the VP7 genes of a) strain D and other serotype 1 human rotaviruses, b) strain DS-1 and other serotype 2 human rotaviruses, c) strain P and other serotype 3 human rotaviruses; and d) strain ST3 and other serotype 4 human rotaviruses. However, hybrid bands could not be demonstrated between the VP7 genes of D, DS-1, P, or ST3 and human rotaviruses belonging to a different serotype. This technique was also used to examine RNA extracted from stools of children hospitalized with rotavirus diarrhea. All five viruses with a "short" RNA pattern shared homology with the DS-1 strain VP7 gene; two of them had been previously adapted to tissue culture and shown to be serotype 2 strains by neutralization. Of the remaining 10 viruses, with "long" RNA patterns, two hybridized to the D strain VP7 gene, six hybridized to the P strain VP7 gene, and two hybridized to the ST3 strain VP7 gene. Hybridization using single human rotavirus gene substitution reassortants as probes may provide an alternate way of serotyping field isolates and would circumvent a need for tissue culture adaption.